Structured illumination has been used to substantially enhance localization precision in microscopy. Hausmann et al (U.S. Pat. No. 7,342,717 B1) have used a standing-wave interference pattern in the observation axis between two microscope objectives to measure both phase and amplitude of fluorescence excitation by moving the objects in the direction of observation (Z-axis) through the fluorescence excitation standing-wave field and observing fluorescence emission. By making use of the information both from the phase and the amplitude of the standing wave field, not only object-to-object localizations in the Z-axis (i.e. along the optical axis or the axis of observation) could be measured with a precision of some ten nanometers but also object size estimations in the range of 20 to 150 nm were obtained, previously not possible using conventional light microscopy.
Successful attempts to use laterally structured illumination, i.e. illumination structured in the plane orthogonal to the direction of observation have been reported (Reto Fiolka et al, OPTICS LETTERS, Vol. 33, No. 14, 1629-1631, 2008). A method combining structured illumination in the plane of observation with total internal reflection fluorescence illumination (TIRF) has been disclosed in U.S. Pat. No. 6,987,609 B2.
A drawback of previously disclosed methods to feed the interfering light beams into the observation plane (i.e. plane orthogonal to the direction of observation or X-Y-plane) for TIRF microscopy is that they are complicated. Thus for example, if small prisms or mirrors are used within the microscope objective area, the mechanical adjustment needed to control the critical angle for total internal reflection (TIRF) excitation and the wavelength of the standing-wave field is difficult due to the necessary miniaturization. The same holds true when using fiber optics to insert the interfering light into the microscope objective area. Another drawback is the partial light blocking when inserting devices into the light path of the objective (see U.S. Pat. No. 6,987,609 B2).
If a grating is used, the period and the orientation of the grating cannot be (easily) adjusted.
In (Euiheon Chung et al, Biophysical Journal, 1747-1757, 2007), another optical layout is described to generate a different standing wave excitation under total internal reflection (TIRF) condition using a beam splitter and laterally adjustable fiber tips to select the TIRF angle.
When using a prism as the specimen surface, the adjustments are somewhat less complicated, but here the sample has to be applied directly onto a surface of the prism used to generate the total internal reflection fluorescence excitation.